Confluent human umbilical vein endothelial cells were treated with diamide, t-butyl hydroperoxide (t-BH) or the hydrogen peroxide generating system glucose/glucose oxidase and the effects on glutathione oxidation and protein S-thiolation were examined. In the presence of all three oxidants glutathione was rapidly oxidized to a similar extent and S-thiolation of a limited number of proteins occurred. Diamide caused considerable S-thiolation of proteins with molecular masses of 44, 34, 24 and 14 kDa, of which the protein with molecular mass of 44 kDa was most extensively modified. t-BH caused extensive modification of proteins with molecular masses of 24 and 14 kDa whilst hydrogen peroxide caused S-thiolation of proteins of 39, 24 and 14 kDa. This study shows that S-thiolation of proteins is an important metabolic response to oxidant insult in human endothelial cells and that the specificity of the response depends on the chemical nature of the oxidant.